The long-term objective is to target antigens encoded by cancer-specific somatic mutations by adoptive immunotherapy with TCR-transduced T cells. Such antigens are truly cancer-specific and mostly caused by non-synonymous base substitutions that alter a single amino acid in a protein. Each cancer harbors a unique set of mutations and antigens. Cancer patients may harbor TCRs that recognize mutant peptides encoded by these mutations, but T cells bearing these receptors are often energized or tolerized. This proposal explores the use of adoptive transfer of T cells transduced with mutation-specific TCRs. Under ideal conditions, a single transduced TCR (1D9) can eradicate large established cancers while targeting only a single mutant antigen (mp68). This peptide binds MHC with sub-nM affinity. However, if this antigen is lost the cancer may escape. Furthermore, the TCRs and mutant antigens found in some cancer patients may not have the qualities needed to eradicate their cancer. Aim 1 sets out to compare the quality and diversity of mp68-specific TCRs isolated from tumor-free immunized mice or from mice at various stages of tumor growth. The prototype mp68 antigen and the 1D9 TCR will be used to test whether TCRs of tumor-eradicating potential can not only be isolated from tumor-free immunized mice but also from malignant peritoneal exudate or tumor-infiltrating lymphocytes (TILs) of tumor bearing mice. Isolating TCRs from single T cells in a cancer-specific T cell population will make time-consuming derivation of T cell clones superfluous. Furthermore, it will be tested whether immunocompetent mice bearing a progressively growing mp68+ tumor have antigen-specific but anergized T cells, yet their TCRs can eradicate the mp68+ cancer when transduced into non-anergized PBL of the tumor bearer. Aim 2 is to determine the ability of TCR-transduced T cells to destroy UV-induced cancers that grow aggressively in immunocompetent mice despite retaining many mutant antigens, including some encoded by driver mutations. It will be tested whether a combination of different TCRs targeting a set of independent unique antigens can eradicate large established cancers and/or whether co-transfer of CD8+ with CD4+ TCR-transduced mutation-specific T cells that recognize mutation peptide presented by tumor stroma prevents escape of antigen-loss variants (ALVs). Spontaneous mouse tumors and most human cancers harbor ~10x fewer mutations than UV- or MCA-induced cancers in mice or UV- or tobacco-induced melanoma and NSCLC in humans. Therefore, Aim 3 is to determine the therapeutic potential of TCR-transduced T cells against a spontaneous mouse tumor and human ovarian cancer. Despite having one log fewer mutations, both cancers still encode multiple mutant peptides of an pMHC affinity that could be sufficient to achieve tumor destruction, but might not prevent escape of AVLs unless multiple epitope are targeted. Mutation-specific TCRs will be isolated from short-term-expanded T cell cultures of TILs or malignant effusions. TCR-transduced T cells will be used in vitro to identify mutant antigens that cannot be lost from the cancer cell and serve as better targets.